IGS ncRNAs are increased in human cells with SETX deficiency and rescued by human RNaseH1 expression. (A) Schematic diagram of a single copy of human rDNA cassette (∼43 kb) with locations of primer pairs used for reverse transcription with quantitative polymerase chain reaction (RT–qPCR). (B) Total RNA was extracted from U2OS cells with control or SETX shRNA. IGS ncRNAs were analyzed by RT-qPCR with primers as indicated in A and quantified in three independent experiments, normalized to levels of 7SK control RNA. (C) Total RNA was isolated from SETX-depleted U2OS cells with control or human wild-type RNaseH1 overexpression, and the levels of IGS ncRNAs were measured and quantified as in B. (D) RNA extracted from U2OS control or SETX knock-out cells was probed by northern blotting for IGS18 and IGS22 ncRNAs, with 7SK as an internal control. (E) Total RNA was extracted from U2OS cells with SETX knock-out and human RNaseH1 overexpression. IGS ncRNAs were analyzed and quantified by RT-qPCR and normalized to 7SK control gene as in B. (F) Total RNA was extracted from U2OS control or SETX knock-out cells and measured by TGIRT-seq. The peaks of IGS ncRNAs were visualized by IGV software. Top panel: positive-strand reads; bottom panel: negative-strand reads. Locations of repeats related to Alu elements as well as long repeat 1 and 2, Sal X 3, the CDC7 pseudogene, and the 3ʹ end of the PAPAS lncRNA are shown. (G) R-ChIP accumulation in the IGS region in WT and SETX KO U2OS cells. Error bars in B, C, and E indicate standard deviation. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001, and 0.0001, respectively, by two-tailed t test assuming unequal variance between control and SETX-depleted groups, or between SETX-depleted cells with control or human RNaseH1 overexpression. Source data are available for this figure: SourceData F8.